Antimicrobial Peptides From Myna Birds PDF

i INTRODUCTION Antimicrobial resistance presents a significant challenge in the field of infectious diseases, as pathogens develop mechanisms to evade traditional antibiotics. As antibiotics have been used and abused so extensively in both human medicine and agriculture over the past century, this...

i INTRODUCTION Antimicrobial resistance presents a significant challenge in the field of infectious diseases, as pathogens develop mechanisms to evade traditional antibiotics. As antibiotics have been used and abused so extensively in both human medicine and agriculture over the past century, this has expedited the development of antibiotic resistance and become a serious worldwide health problem (English & Gaur, 2009). As a result of this problem that threatens public health, it has become necessary to apply intensive research in finding alternative conventional antibiotics. Antimicrobial peptides (AMPs) are small cationic peptides that are present in many species with great potential. These naturally occurring peptides are essential to the innate immune systems of many different species, including humans. Because of their capacity to battle a variety of infectious pathogens, including bacteria, viruses, mycobacteria, fungi, and even some parasites, they have attracted a great deal of attention in the fields of medicine and research (Narayana & Chen, 2015). They are generated by the phagocytic and mucosal epithelial cells of the host (Lee et al., 2016). Cathelicidins are multifunctional peptides with antimicrobial activity and immunomodulatory functions that contribute to the host's defense mechanisms (Kai- Larsen & Agerberth, 2008). As members of the AMPs family, they have gained attention for their role in host defense and disease resistance across various species (Na et al., 2015, and Nijnik et al., 2012), including Gram-positive and Gram-negative bacteria, fungi, and even drug-resistant strains (Luo et al., 2019, and Schauber et al., 2006). Owing to their invasive and scavenging lifestyle, Common Myna Birds (Acridotheres tristis) are likely have important mechanisms, including AMPs, to resist infection. This study focuses on the identification and characterization of the Cathelicidin from Acridotheres tristis, with antibacterial activity. For structural analysis and antibacterial property prediction, bioinformatics tools and databases were employed. Potential peptides were synthesized in an effort to validate these predictions and in vitro assays for antimicrobial activity were conducted. 1  1. LITERATURE REVIEW 1.1. Antibiotic Resistance The 1930s witnessed the development of antibiotics, which brought in one of the most advantageous times in medical history. Penicillin's discovery by Alexander Fleming and the subsequent commercialization of this and other families of chemicals are thought to have contributed to a 10-year rise in average life expectancy (Mcphee & Hancock, 2005). Antimicrobial drugs, specifically antibiotics, are medications that either destroy or inhibit the growth of bacteria, they have been used extensively in the treatment of many human diseases to treat a wide range of bacterial illnesses, saving many lives. However, because of the extensive usage of antibiotics in agricultural, veterinary, and human healthcare bacteria have evolved resistance mechanisms over time, and many antibiotics are now either completely worthless or ineffective. As a result of this process, multidrug-resistant bacteria also known as superbugs have emerged and become a serious threat to public health (Moretta et al., 2021 and, Geisinger & Isberg, 2017). In addition, inadequate care means that if the antibiotic treatment is not taken probably the bacteria may develop resistance (Espinosa, 2009). Moreover, one of the natural changes that make microorganisms adapt requires evolution which develops antibiotic-resistant mutations (Rodríguez-Rojas et al., 2013). Also, bacteria can propagate resistance by passing resistance genes to one another by a process called horizontal gene transfer (Huddleston, 2014). This resistance highlights the emergency to develop alternative therapeutic agents. 1.2. Antimicrobial peptides and their significance The antibacterial activities of antimicrobial peptides were identified by Spitznagel and Zeya in the 1960s. After Boman noted that Drosophila melanogaster continued to live even after being injected with both virulent and non-virulent strains of Aerobacter cloacae, AMPs became the subject of extensive research beginning in 1972. This observation highlighted the potential of AMPs in treating bacterial illnesses (Pasupuleti et al., 2011). AMPs are also alternately known as host defense peptides (HPDs) as they provide protective barriers in innate immunity against microbial invasion, including bacterial, viral, and fungal infections. AMPs are effective against a variety of diseases due 2 to their broad-spectrum antimicrobial activity and operate as the first line of defense against invasive pathogens, in contrast to antibiotics, which are usually restricted to particular bacterial species (Wang et al., 2016, Bowdish et al., 2005 and Mcphee & Hancock, 2005). These peptides can be produced in tissues throughout the body including the epidermis, mucosal surfaces, and immune system cells. They may be generated in response to an immunological stimulus, an infection, an inflammation, or be constitutively present. Following the translation of these peptides, they become active by post-translational modifications (Sahl et al., 1995). They often have an amphipathic structure, i.e. both hydrophilic (as a water lover) and hydrophobic (which water hates) areas. These peptides usually carry a positive charge that enables them to interact with pathogens' negatively charged surfaces, such as those of bacteria and fungus, and allow breaking their cell membranes and ultimately destroying them (Figure 1). When microbial cell membranes are disrupted, the microbe may leak internal materials, experience cell lysis, or even die (Yeung et al., 2011). Figure 1: The mechanism of action for antimicrobial peptides against microorganisms where the positive charge represented in the peptides is attracted to the negative charge in the pathogens and interacts until it leads to killing the microbe. 3 Antimicrobial peptides' capacity to target conserved components on the microbial surface, such as teichoic acids in Gram-positive bacteria or lipopolysaccharides in Gram-negative bacteria, is thought to be responsible for their ability to combat a variety of pathogens (Li et al., 2014). Apart from their antibacterial characteristics, AMPs serve crucial roles in the immune system by affecting the synthesis of cytokines and chemokines, signaling molecules involved in immune cell recruitment and activation, and regulating the inflammatory response (Duarte-Mata & Salinas-Carmona, 2023). Moreover, it has been demonstrated that AMPs exhibit wound-healing abilities and regeneration of injured tissues, through stimulating cell migration and proliferation, encouraging the creation of new vessels of blood (angiogenesis), and increasing the synthesis of extracellular matrix components (Figure 2) (Vijayan et al., 2019). Figure 2: General characteristic of antimicrobial peptides as part of the innate immune system The antimicrobial peptides are also being researched for their possible application in the management of autoimmune and inflammatory diseases (Zhu et al., 2022). 4  1.3. Characteristics AMPs have many distinctive features compared to traditional antibiotics, including broad- spectrum activity where AMPs can selectively kill a wide range of pathogens, including antibiotic-resistant bacteria (Gottlieb et al., 2008). Also, they are useful in treating infections caused by a wide range of pathogens, such as bacteria, viruses, fungi, and parasites, this includes bacteria that are Gram-positive or Gram-negative, and drug- resistant types that are immune to traditional antibiotics (Chen et al., 2018). In addition, these peptides can be used in combination therapies to increase the effectiveness of conventional antibiotics and lower the dosage needed while minimizing the emergence of resistance (Jorge et al., 2017). As a possible alternative to conventional antibiotics, researchers are looking at the medicinal potential of AMPs. For external use, AMPs can be applied to prevent and cure localized infections, such as creams, ointments, and wound dressings (Park et al., 2010). These peptides have minimal chance of resistance development as they have a complicated mode of action compared to conventional antibiotics, which put bacteria under selection pressure and can cause resistance to grow. They cause bacterial membranes to rupture, which hinders the development of bacterial resistance (Wang et al., 2018). Furthermore, an investigation into medicinal uses where the current studies are looking at the application of AMPs in several clinical aspects, such as respiratory infections, wound healing, and replacement of traditional antibiotics (Kang et al., 2016). Low likelihood of resistance where AMPs usually cause disruption to microbial cell membranes or have complex modes of action, in contrast to standard antibiotics that frequently target particular components of bacteria. Pathogens find it more difficult to evolve resistance as a result (Wang et al., 2018). One of the promising features of antimicrobial peptides is the Possibility of peptide engineering, scientists can modify AMPs to improve their antibacterial qualities, stabilize them better, and lessen their toxicity. This creates opportunities for the creation of new anti-infective drugs (Lei et al., 2021). Moreover, AMPs have a wide variety of diverse sequences and structures, which helps them fight against different infections (Figure 3). Examples of common structural 5 motifs in AMPs are Beta-sheets, cyclic peptides, and alpha-helices (Koehbach & Craik, 2019). Figure 3: Secondary structures antimicrobial peptides Antimicrobial peptides can disrupt the biofilms of bacteria. They have demonstrated potential in uprooting the intricate structures known as biofilms that stow away germs from both the immune system and antibiotics. They are therefore useful in the treatment of persistent infections (Sedarat & Taylor-Robinson, 2022). In addition to the mechanisms of action to eradicate infections, AMPs use a variety of strategies. They can damage microbial cell membranes, obstruct intracellular functions, or target certain chemicals found in infections (Bechinger & Gorr, 2016). Certain AMPs have the ability to modify the host's immunological response or immunomodulatory effects. Infection control and 6 inflammation reduction may benefit from this capacity (Banas et al., 2008). Moreover, with innate evolutionary protection over millions of years, antimicrobial peptides have developed into a component of the body's natural defensive systems. In the battle against diseases, they symbolize a sustainable and natural resource that is in line with the concepts of eco-friendly medicine. Creative approaches are required to address the danger of antibiotic resistance to global health. These natural peptides have shown promise in the fight against diseases resistant to antibiotics. Antimicrobial peptides in combination with anti-infective agents present a number of challenges and considerations, such as toxicity (since some AMPs can be toxic at high concentrations and safety profile optimization is essential), stability (because AMPs can degrade under certain circumstances, such as in the presence of enzymes), and the need to develop efficient delivery strategies to guarantee that AMPs reach the infection site (De Oliveira et al., 2023). An intriguing and exciting family of anti-infective medicines is antimicrobial peptides. Their potential for several uses, low probability of resistance, and broad-spectrum efficacy provide them an invaluable asset in the fight against infectious illnesses. In order to make them safer and more effective for use in therapeutic settings, ongoing research is concentrated on maximizing their potential while overcoming obstacles. 1.4. Antimicrobial Peptides Classification Antimicrobial peptides are typically short, consisting of 10 to 100 amino acids. They exhibit multiple mechanisms of action, which makes it difficult for pathogens to develop resistance against them (Figure 4) (Zhang et al., 2021). They are categorized into different families based on their origin and structural characteristics. The primary families include defensins, cathelicidins, and histatins. Defensins are further divided into α-defensins and β-defensins, found in neutrophils and epithelial cells, respectively. Cathelicidins, such as LL-37, are stored in neutrophil granules and released upon activation. Histatins are primarily found in saliva and play a crucial role in oral immunity. Each family of AMPs has evolved to target specific pathogens and is integral to the innate immune response. These families not only provide direct antimicrobial action but also modulate the host's immune system (Talapko et al., 2022). 7  Figure 4: Several ways to classify antimicrobial peptides (AMPs) The structure of AMPs is integral to their function, generally amphipathic, and has both hydrophilic (water-attracting) and hydrophobic (water-repelling) regions. This amphipathic nature allows them to interact with and disrupt microbial membranes. Based on structure, AMPs can be classified into several structural types: α-helical peptides, β-sheet peptides, and extended or looped peptides. α-helical peptides, such as magainins, adopt a helical structure in the presence of membranes. β-sheet peptides, like defensins, contain β-strands connected by disulfide bridges, providing stability and rigidity. Extended peptides, such as indolicidin, lack a defined secondary structure but are rich in specific amino acids like tryptophan and proline (Mabrouk, 2022) They are typically rich in cationic amino acids like lysine and arginine, which contribute to their positive charge. This positive charge facilitates the initial electrostatic attraction between the peptide and the negatively charged microbial membranes. Hydrophobic 8 amino acids, such as tryptophan and phenylalanine, enable the peptides to insert into and disrupt the lipid bilayer of pathogens. The specific sequence and composition of amino acids in AMPs determine their structural conformation and, consequently, their antimicrobial efficacy. The balance between cationic and hydrophobic residues is essential for the optimal activity of AMPs (Huan et al., 2020). In summary, AMPs employ multiple mechanisms to exert their antimicrobial effects. The primary mode of action involves the disruption of microbial membranes. Upon binding to the membrane, AMPs can form pores or channels, leading to membrane permeabilization and cell lysis. Some AMPs translocate across the membrane and interact with intracellular targets, disrupting vital processes such as protein synthesis, DNA replication, and enzyme activity. Additionally, AMPs can modulate the host immune response by enhancing the recruitment and activation of immune cells, promoting wound healing, and reducing inflammation. This multifaceted mode of action makes AMPs highly effective against a wide range of pathogens and reduces the likelihood of resistance development (Seyfi et al., 2019). 9  1.5. Myna bird The Indian common myna bird (Acridotheres tristis), originally from southeast Asia, was introduced to many places in the world for different reasons, including insect control (Figure 5). Figure 5: The general appearance of the Myna bird, which is found in some areas around the world. (Griffin, P..2011) Due to their omnivorous diet, ability to adapt to a variety of environments, and aggression against other birds and small mammals, these species have quickly expanded throughout the world. Mynas are now considered to be among the top 100 invasive pest species worldwide as a result (Lowe et al., 2011). Myna bird was first reported in Oman in the Muscat Governorate in 1982 (Desmond, 2022). Since then, the population of myna birds has sharply increased. The Environment Authority is working with specialists to develop solutions and is exerting every effort to reduce the population of this invasive bird (Nassriya, 2022). The ecological significance of invasive mynas makes them interesting to study from the perspectives of population dynamics, biology, aggressive behavior, and potential vectors for known and novel infectious agents. 10 They are most suited to urban settings that have been broken up by human settlements, and they particularly like open habitats in tropical and subtropical regions. As omnivorous, opportunistic feeders, they consume a variety of foods, including pet food, tiny reptiles, cereal crops, ripening papaya, banana, fig fruits, and invertebrates. Pathogens such as Trichomonas gallinae, Haemosporidian parasites (Plasmodium and related Haemoproteus spp.), Salmonella spp., and mites have also been demonstrated to be capable of being carried by invasive mynas (Clark et al., 2015). While low pathogenic avian influenza and avian siadenovirus were found in a prior investigation on free-living mynas, nothing is known about the clinical and zoonotic hazards associated with whatever viruses these animals may carry (Fierer et al., 2017). Owing to their invasive and scavenging lifestyle, Common Myna (Acridotheres tristis) likely have AMPs, to resist infection. 1.6. Cathelicidins Identification Cathelicidins are a fascinating family of antimicrobial peptidesAMPs found across various vertebrates, playing a crucial role in innate immunity (Tossi et al., 2024). They are identified based on their unique structure, which includes the signal peptide that directs the peptide to the secretory pathway (Choi et al., 2022). Also, the cathelin domain the is a conserved proregion and a highly variable antimicrobial domain that becomes active upon proteolytic release (Zanetti, 2003). Cathelicidin identification in different organisms shows variation in amino acid sequence and structure, in mammals, cathelicidins are well studied, some examples like LL-37 in humans, CRAMP in mice, and BMAP-28 in cattle (Khurshid et al., 2017). Reptiles and Fish exhibit a more complex arrangement of cathelicidin genes, with multiple genes identified in species like crocodilians, snakes, and certain fish (Alford et al., 2020). In birds, cathelicidins are crucial for their immune defense and they typically have a single cathelicidin cluster, with the number of genes varying by species. For instance, chickens have several cathelicidin genes, such as CATH1, CATH2, CATH3, and CATH-B1. Also, in ducks the identified cathelicidins include AvBD-1 and AvBD-2, which are part of the 11 broader avian beta-defensin family but share functional similarities with cathelicidins (Cheng et al., 2015). In general, cathelicidin shows structural similarities to mammalian peptides but are adapted to the specific needs of avian species (Khurshid et al., 2017). The functional insights of cathelicidins in birds are particularly interesting due to their adaptation to avian physiology and immune challenges. These peptides help birds combat a wide range of pathogens, contributing to their overall health and survival. Additionally, they play roles in modulating the immune response, wound healing, and maintaining gut health (Tossi et al., 2024). 12  2. METHODS 2.1. Protein Identification The Myna cathelicidin's gene identification process begins with well-characterized orthologs, the four different types of chicken cathelicidins (CATH-1, -2, -3, and -B1). The gene database at NCBI (https://www.ncbi.nlm.nih.gov/ )was accessed on December 23, 2023, to retrieve the chicken cathelicidin orthologs gene and protein data (Table 1). Table 1: Chicken cathelicidin orthologs gene and protein data Name/Gene Description Location Aliases accession ID number Chromosome 2, CATH- CATH1 cathelicidin-1 NC_052533.1 1, NP_001001605 [Gallus gallus (4015652..4017138, CATH-.1 ID:414337 (chicken)] complement) 3, CATHL 1 CATHB1 Cathelicidin Chromosome 2, CATH- ID:1008584 B1 [Gallus NC_052533.1 B1, NP_001258101 12 gallus (4013411..4014539, cathelici.1 (chicken)] complement) din-B1 CATH2 Chromosome 2, CAMP, ID:420407 cathelicidin-2 NC_052533.1 CATH- NP_001020001 [Gallus gallus (4011425..4013199, 2,.3 (chicken)] complement) CATHL 2, CMAP2 7 CATH3 Chromosome 2, NP_001298106 ID:1008583 cathelicidin-3 NC_052533.1 CATH-.1 43 [Gallus gallus (4009501..4010761) 3, (chicken)] CATHL 3 13 To identify the potential myna bird orthologs, the following steps were performed. First, the RefSeq FASTA sequence for the protein was selected to perform the BLASTp search. Also, in the BLAST NCBI website (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on: December 2023). To identify potential cathelicidins in Myna, we searched for available genomic data, including the Myna bird's annotated sequences (Acridotheres tristis), BioProject accession number PRJNA928557, and the Illumina whole genome resequencing data are available under the BioProject accession number PRJNA1054049. Genome assembly is available on NCBI accession GCA_027559615.1 (Stuart et al., 2024). The whole genome shotgun contigs (wgs) were selected as a database, and the scientific name for the myna bird (Acridotheres tristis (taxid:279927)) as an organism. As the best chicken cathelicidin hit based on the length and present identity was not full, we tried to get the full sequence by identifying the closest peptide ortholog by BLASTp using this fragment (>YTQALAQAVDSYNQRPEVQNAFRLLSAEPEPAPGVELSSLQGLNFTMMETDC AASARRDPEDCDFKENG). This identified an ortholog from Sturnus vulgaris with 96.86% identity. Next, to retrieve the full predicted peptide sequence we run tblastn against the Acridotheres tristis (taxid:279927) wgs. The alignment results for the full sequence from Myna cathelicidin were checked if four fragments were found, representing the number of expected exon regions. 2.2. Conserved Domain Search Service (CD Search) To find out the conserved domain for a specific protein sequence the protein sequence was entered in FASTA format in the NCBI Conserved Domain Search toolbox, (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi, accessed on: December, 2023). The conserved domains that match the sequence were provided in the list of domain hits and the most specific hit selected (usually the top bar). The search tool shows the name, accession, and description for known domains that contain protein as well as the Pssm- ID, Cd Length, Bit Score, and E-value. 14  2.3. Enzyme cleavage using peptide cutter Enzyme cleavage using peptide cutter from Expasy database (https://web.expasy.org/peptide_cutter/, accessed on: December 2023) is a tool that predicts potential cleavage sites cleaved by proteases or chemicals in a given protein sequence (Purcell et al., 2023). PeptideCutter can help to predict the cleavage sites of AMPs by different enzymes, such as neutrophil elastase, which is involved in the inflammatory response and the defense against pathogens (Song et al., 2018). By using PeptideCutter (https://web.expasy.org/peptide_cutter/, accessed on: December 2023). The identified protein sequence is pasted in the search box in row format (MLSSWLLVLAVLGGACALPAPAPLAYTQALAQAVDSYNQRPEVQNAFRLLSA EPEPAPQGVELSSLQGLNFTMMETDCAASARRDPEDCDFKENGAIKECSGPVKI LQGSPEIDLRCSDASSDPVLIQRGRFGRFLGKIRRFRPRVKFDLRLKGSVGLG) and from the section of enzymes list (Neutrophil elastase) and the option of (only the following selection of enzymes and chemicals) need to be selected before submitted the data as well as the way of result need to be displayed, so the display options for the cleavage sites, such as map, table, or probability is chosen. The tool shows the possible cleavage sites and the fragments of the AMP. 2.4. Signal peptide Detecting the signal peptide using SignalP-5.0 is a tool that predicts the presence and location of signal peptides and their cleavage sites in proteins from different domains of life. Signal peptides are short amino acid sequences that direct proteins to their proper cellular compartments, such as the plasma membrane, the endoplasmic reticulum, or the extracellular space (Ghasemi et al., 2022). The benefit of finding out the signal peptide using SignalP-5.0 for specific protein sequences is to identify potential antimicrobial peptides (AMPs). AMPs are usually secreted or membrane-bound proteins that require signal peptides for their proper sorting and activation (Garcion et al., 2021). The amino acid sequence in FASTA format is pasted in the SignalP-5.0 tool (https://services.healthtech.dtu.dk/services/SignalP-5.0/, accessed on: December 2023). Before submitting the sequence (the organism group that the protein belongs to (Eukarya, 15 Gram-positive, Gram-negative, or Archaea)), as well as (the output format for the prediction results (long or short)) should be selected. 2.5. Amp activity prediction The benefit of finding out the AMP activity prediction using a database of Antimicrobial Activity and Structure of Peptides for a specific protein sequence is to discover new antimicrobial peptides (AMPs) or optimize existing ones (Wang et al., 2022). DBAASP (https://dbaasp.org/home, accessed on: December 2023) is a database of antimicrobial activity and structure of peptides that provides information and analytical resources for designing antimicrobial compounds with a high therapeutic index (Pirtskhalava et al., 2020). The amino acid sequence in FASTA format was pasted in the DBAASP tool (https://dbaasp.org/tools?page=property-calculation, accessed on: December 2023), and the sequence was submitted the results will appear in the table. Note that the full sequence cannot be included fully only the sequence which is used is after the cleavage position at the end of the protein sequence obtained from enzyme cleavage using a peptide cutter from the expasy database 1.1. Structure using HeliQuest and RCSB PDB The benefit of finding out the structure of the antimicrobial peptides using HeliQuest and RCSB PDB for a specific protein sequence is to design and optimize new AMPs with high helicity and amphipathicity. Researchers can determine whether a peptide sequence has a strong amphipathic character and a high helical propensity—two characteristics essential to the antibacterial action of AMPs—by utilizing HeliQuest. Additionally, examines the effects of varying peptides' helical and physicochemical characteristics on their antibacterial and cytotoxic qualities. Additionally, create new AMPs or alter current ones by altering the peptide's length, orientation, or content of amino acids (De Cena et al., 2022). RCSB PDB (https://www.rcsb.org/, accessed on: December, 2023) used in order to predict the secondary structure of the Myna-Cath peptide. The peptide sequence (VLIQRGRFGRFLGKIRRFRPRVKFDLRLKGSVGLG) is pasted in the RCSB PDB web page and then it was submitted. HeliQuest (https://heliquest.ipmc.cnrs.fr/, accessed on: December, 2023) is a web server that calculates the helical and physicochemical 16 parameters of a given peptide sequence and allows the user to screen peptide databases for helical AMPs (Huan et al., 2020). The peptide sequence and then it was submitted, note that only the amino acid sequence, i.e. to FASTA format, was used in this tool. 1.2. Multiple sequence alignment (MSA) and phylogenetic tree Multiple sequence alignment (MSA) is a process of aligning group protein sequences based on their similarity and evolutionary relationships. The phylogenetic tree is a graphical representation of the evolution of a group of organisms or genes (Riley et al., 2023). Using the BLAST tool at the UniProt database https://www.uniprot.org/, accessed on: December 2023), which is a comprehensive resource of protein information, MSA and phylogenetic tree were generated for the predicted protein sequence (Irajie et al., 2016). Briefly, the analysis was restricted by taxonomy (Aves [birds] 8782) and sequences of similar protein sequences (orthologs) in the UniProt database were selected. All selected subjects were aligned with the query, using the align tool to generate the MSA and the phylogenetic tree. The same method through running blast in UniProt done for full protein sequence is performed to peptides sequence that has been obtained from enzyme cleavage using peptide cutter form expasy database (>Myna_peptides VLIQRGRFGRFLGKIRRFRPRVKFDLRLKGSVGLG), as in the restrict by taxonomy (Aves [birds] 8782) is selected then run blast. The peptide sequence of similar sequences for other orthologues in the UniProt database is selected. All these selections were aligned with the query, an appropriate alignment algorithm and parameters to generate the MSA of the query sequence with the selected sequences were chosen and then an alignment button was selected, on the top page of the result, the tree option was chosen to view the phylogenetic tree. 1.3. Peptide Synthesis In order to obtain the identified antimicrobial peptide for the characterization test, the peptide sequence submitted to GL Biochem (Shanghai) Ltd. (http://www.glschina.com/en/ser/peptide/custompeptide.htm ) 17  1.4. Bacterial Strains The selective bacteria included in this study were Pseudomonas aeruginosa (ATCC 27853), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Klebsiella pneumonia (ATCC 1706) negative for the blaKPC gene, Klebsiella pneumonia (ATCC 1705) carbapenemase (KPC) producer, and Methicillin-Resistant Staphylococcus aureus (ATCC 700699). 1.5. Inhibition Zone assay Bacteria were streaked from a stock solution on LB agar using a sterile loop, the bacteria were gently spread over a section of the plate to create the first streak. The plate was incubated overnight at 37°C. The next day, three single colonies of bacteria were added to 6 ml of Luria Broth (LB) media, and the mixture was incubated while shaken (using a Stuart Rotator TR-2) for two hours at 37°C in an incubator (Gallenkamp Model INC.200.210C Plus Series Incubator, England). A spectrophotometer (Thermo Electron Corporation Helios Beta Spectrophotometer Model 9423 UVB 133214, England) was used for measuring the bacterial optical density (OD). Once it reached OD 0.6, the bacteria were diluted with LB media at a ratio of 1:100. Next, 6 ml of LB media with 1% agarose already on the water path at 50°C–55°C was mixed with 50 µl of the dilution, put onto a 90 mm petri dish 1 mm thick, and allowed to solidify for 30 minutes. To test the peptide treatments (200µM, 100µM, 50µM, 25µM, and 12.5µM), 3 µl of each were placed into small wells with a diameter of 3 mm. PBS was utilized as a negative control. All plates were incubated overnight at 37°C, and the diameter of the microorganisms' free zone was determined in centimeters (Al Adwani et al., 2020). 4.11. Colony Forming Units (CFU) assay Bactria were streaked from a stock solution on LB agar using a sterile loop, the bacteria were gently spread over a section of the plate to create the first streak. The plate was incubated overnight at 37°C. The next day, three single colonies of microbes were placed in 6 ml of LB media, and the mixture was shaken for two hours at 37°C in an incubator (Gallenkamp Model INC.200.210C Plus Series Incubator, England) using a Stuart Rotator 18 TR-2. After adjusting the O.D. to 0.1, 90 µl of the culture and 10 µl of each of the peptide treatments (200µM, 100µM, 50µM, 25µM, and 12.5µM) were added to 96-well plates as duplicates. PBS was used as a negative control. The plates were then incubated for three hours at 37°C (Incu-ShakerTM Shaking Incubator, Fisherbrand). Next, PBS was used to create a series of dilutions of the incubated culture, resulting in 1:1–10 dilutions. Afterward, 25 µl was spotted on LB agar plates and incubated at 37°C overnight. Lastly, the CFU of microorganisms was calculated using the formula (CFU= (number of colonies X Dilution Factor) / Volume Plated) (Al Adwani et al., 2020). 4.12. Scanning Electron Microscope (SEM) SEM was performed based on (Al-Farsi et al., 2019), with some modifications, to assess the impact of the synthesized peptide at different concentrations (50 µM–100 µM) on the same family of bacteria using two different stains, sensitive and resistant (K.pneumonia ATCC 1706 and K.pneumoniae ATCC 1705), respectively. Briefly, one milliliter of either treated or untreated bacterial broth and 0.5 milliliters of EM 2.5% Karnovsky & fixative were put into an Eppendorf tube, re-suspended, and kept at 4 c overnight. Then centrifuged for 5 minutes at 5,000 rpm. The supernatant was disposed of and the pellets were re- suspended for 10 minutes in 1 milliliter of sodium cacodylate washing buffer, centrifuged for 5 minutes at 5,000 rpm. After discarding the supernatant, the pellets were fixed with 1 milliliter of 2% osmium tetroxide and dehydrated with a range of alcohol percentages beginning with two washes of distilled water, 25% ethanol, 75% ethanol, 95% ethanol, and finally two washes of 99.9% ethanol. Subsequently, 1 milliliter of a 1:1, v/v hexamethydisalizane (HMDS)/ethanol combination was added and allowed to settle for half an hour. After that, another 1 milliliter of HMDS was added and left for 20 minutes. Following that, samples were treated with pure HMDS and allowed to dry for three hours at room temperature. Later, dried bacteria samples were placed within 10 mm aluminum stubs and sealed with double-sided carbon adhesive. Afterward, they were observed with a Jeol JSM-5600LV scanning electron microscope (Japan) after being coated with gold particles using a BioRad coating system (BIO-RAD, Microscience Division Serial No. 88091, England). The bacteria samples' topographic micrographs have been obtained and stored as pictures. For every strain, several independent SEM analyses were carried out. 19  4.13. Statistical Analysis For inhibition zone assay and colony forming unit assay, the analyses were performed in three independent experiments with duplicates and presented as mean values ± and standard deviation. GraphPad Prism version 10.3.0 (GraphPad, La Jolla, CA), was used to analyze the data and create the graphs. The data was first tested for normality and then one-way ANOVA was applied for the normally distributed data. Multiple unpaired t-tests were applied to compare K. pneumonia ATCC 1706 and K. pneumoniae ATCC 1705. The P-value of VLIQRGRFGRFLGKIRRFRPRVKFDLRLKGSVGLG). Based on the activity prediction the normalized hydrophobic moment to understand the peptide interaction with the biological membrane is 0.22 which indicates a moderate level of amphipathicity in peptide structure. The normalized hydrophobicity is -0.55 this value indicates the peptide tendency towards hydrophilicity. The minimum net charge to be considered effective falls with (+2 to +13) and as the predicate activity the identified peptide net charge is considered good as it shows the value of 10. The isoelectric point is 12.59 which indicates the peptide is highly basic and it can interact with (amino acid_ acidic) negatively charged membranes of bacteria more easily. The amphiphilicity index value is 0-94 which is quite high means the peptide has distinct hydrophobic and hydrophilic regions. (Table 4) 28 Table 4: The output result using the antimicrobial peptide activity database to predict the activity for the identified Myna-Cath sequence Normalized Hydrophobic Moment 0.22 Normalized Hydrophobicity -0.52 Net Charge 10.00 Isoelectric Point 12.59 Penetration Depth 16 Tilt Angle 90 Disordered Conformation Propensity -0.12 Linear Moment 0.18 Propensity to in vitro Aggregation 0.00 Angle Subtended by the Hydrophobic 40.00 Residues Amphiphilicity Index 0.89 Propensity to PPII coil 0.94 29  5.2.2. RCSB PDB and HliQust structure prediction The output of utilizing the RCSB PDB to model the putative Myna-Cath peptides to clarify their secondary and tertiary structures (Figure 12). This allows the examination of the possibility of interacting with biological membranes and additional structural components that support the antibacterial activity. Figure 12: Alphafold structural models of putative antimicrobial peptides from the annotated cathelicidins of Acridotheres tristis. The structure is constructed from the RCSB PDB database. PDB DOI: https://doi.org/10.2210/pdb2GDL/pdb The projected model for the peptide's secondary structure included an alpha-helical, according to the predictions. The sequence hydrophobicity and net charge of each peptide can be estimated using HliQust structure prediction, (Figure 13) to determine if hydrophobic amino acids were concentrated on one side of the helix, with polar or hydrophilic amino acids on the other. The result shows the hydrophobic and hydrophilic regions are separated which helps to fold the peptides and create alpha-helical peptides that are commensurate with the cathelicidin structure. This peptide's helical structure makes it possible to determine each peptide's hydrophobicity, net charge, hydrophobic moment, and hydrophobic face directed toward the hydrophobic core. 30  Figure 13: Alpha helices are present in the putative antimicrobial peptides from the annotated cathelicidins of Myna-Cath (Acridotheres tristis), according to the helical wheel structure prediction finding. Yellow-colored residues are hydrophobic, whereas blue-colored residues are cationic. The hydrophobic moment's path is shown by the arrow. 31  5.3. Antimicrobial Activity 5.3.1. Inhibition Zone Assay The antimicrobial activities of Myna Cath-Peptide were tested against several bacterial strains. The highest-sensitive bacteria were Klebsiella pneumonia (ATCC 1706) with an inhibition zone of around 0.83cm, 0.73cm, and 0.63cm at the higher concentration of the peptide at 200µM, 100µM, and 50µM respectively. On the other hand, the inhibition zone was around 0.53cm and 0.41cm at the lowest concentrations 25µM, and 12.5µM, respectively. (Figure 14). Figure 14: Antibacterial activity of Myna-Cath peptide against K. Pneumonia ATCC 1706 was examined by inhibition zone assay, using different concentrations of Myna- Cath peptide at 12.5µM, 25µM, 50µM, 100µM, and 200µM including one negative control using PBS. statistical analysis using one-way ANOVA. The P-value of